The primate lentiviruses human immunodeficiency virus (HIV) type 1 (HIV-1), and type 2 (HIV-2) cause the disease AIDS, and are epidemic in human populations world wide. HIV-1 and HIV-2 are genetically related, antigenically cross reactive, and share a common cellular receptor (CD4). See, Rosenburg and Fauci (1993) in Fundamental Immunology, Third Edition, Paul (ed) Raven Press, Ltd., New York (Rosenburg and Fauci 1) and the references therein for an overview of HIV infection.
The surface glycoprotein CD4 is found mainly on a subset of T cells, monocytes, macrophage and some brain cells. HIV has a lipid envelope with viral antigens that bind the CD4 receptor, causing fusion of the viral membrane and the target cell membrane and release of the HIV capsid into the cytosol. HIV corrupts the cellular machinery of infected cells by causing the cell to replicate copies of the HIV virus, and eventually death of infected immune cells, thereby disabling the immune system and killing the patient due to complications associated with a disabled immune system. HIV infection also spreads directly from cell to cell, without an intermediate viral stage. During cell-cell transfer of HIV, a large amount of viral glycoprotein is expressed on the surface of an infected cell, which binds CD4 receptors on uninfected cells, causing cellular fusion. This typically produces an abnormal multinucleate syncytial cell in which HIV is replicated and normal cell functions are suppressed.
Due to the pandemic spread of HIV, an intense world-wide effort to unravel the molecular mechanisms and life cycle of these viruses is underway. It is now clear that the life cycle of these viruses provides many potential targets for inhibition by gene therapy, including cellular expression of transdominant mutant gag and env nucleic acids to interfere with virus entry, TAR (the binding site for tat, which is typically required for transactivation) decoys to inhibit transcription and trans activation, and RRE (the binding site for Rev; i.e., the Rev Response Element) decoys and transdominant Rev mutants to inhibit RNA processing. See, Wong-Staal et al., PCT/US94/05700, and Rosenburg and Fauci (1993) in Fundamental Immunology, Third Edition Paul (ed) Raven Press, Ltd., New York and the references therein for an overview of HIV infection and the HIV life cycle. Gene therapy vectors utilizing ribozymes, antisense molecules, decoy genes, transdominant genes and suicide genes, including retroviruses are found in Wong-Staal et al., id, and Yu et al., Gene Therapy (1994) 1:13-26. Antisense and ribozyme therapeutic agents are of increasing importance in the treatment and prevention of HIV infection.
In previous studies, the efficacy of several anti-HIV-1 hairpin ribozymes in inhibiting virus replication in human T cell lines was demonstrated. See, Wong-Staal et al., PCT/US94/05700; Yamada et al., Virology (1994) 205:121-126; Yamada et al., Gene Therapy (1994) 1:38-45; Yu et al., Proc Natl. Acadi. Sci. USA (1993) 90:6340-6344, and Yu et al., Virology (1995) 206:381-386. With an anti-U5 ribozyme which targets a highly conserved region of the HIV-1 genome, it was shown that intracellular immunization of primary lymphocytes or hematopoietic progenitor cells lead to resistance of both lymphotropic and macrophage tropic HIV-1 strains (Leavitt et al., Hum. Gene Ther. (1994) 5:1115-1120; Yu et al., Proc. Natl. Acad. Sci. USA (1995) 92:699-703).
Recent studies of the dynamics of HIV replication in patients under antiviral therapy have reaffirmed the central role of the virus in disease progression, and provided a strong rationale for the development of effective, long term antiviral therapy (Coffin, J. M. Science (1995) 267:483-489; Ho et al., Nature (1995) 373:123-6; Wei et al., Nature (1995) 373:117-22). One interesting parameter from these studies is the extremely short life span of an HIV-1 infected CD4.sup.+ lymphocyte (half life=1-2 days), contrasting data from other studies which gave an estimated lifespan of months to years for uninfected lymphocytes (Bordignon et al., Hum Gene Ther. (1993) 4:513-20). These observations are relevant for antiviral gene therapy, because a cell resistant to viral infection, or which suppresses viral replication are strongly selected for in vivo.
Gene therapeutic approaches are hampered by the limitations of the delivery systems currently used in gene therapy. For instance, the extensively used murine retroviral vectors transduce human peripheral blood lymphocytes poorly, and fail to transduce non-dividing cells such as monocytes/macrophages, which are known to be reservoirs for HIV. An appealing alternative would be to utilize HIV-based delivery systems, which would ensure optimal cell targeting and intracellular co-localization of HIV and anti-HIV therapeutics.
Gene therapeutics can target, inter alia, viral RNAs (e.g., using ribozymes, or antisense RNA), viral proteins (RNA decoys, transdominant viral proteins, intracellular single chain antibodies, soluble CD4), infectible cells (suicide genes), or the immune system (in vivo immunization). However, these various gene therapeutic approaches are hampered by the limitations of the delivery systems currently used in gene therapy. For instance, with regard to HIV treatment, the extensively used murine retroviral vectors transduce (transfer nucleic acids into) human peripheral blood lymphocytes poorly, and fail to transduce non-dividing cells such as monocytes/macrophages, which are known to be reservoirs for HIV. An appealing alternative basis for gene therapy vectors would be to utilize HIV-based delivery systems, which would ensure optimal CD4.sup.+ cell targeting and intracellular co-localization of HIV target and gene therapeutic effector molecules. In addition, HIV-derived vectors could be packaged by the wild type virions of patients in vivo and thereby be replicated and disseminated to a larger pool of potentially HIV-infectible cells upon infection by HIV.
Finally, HIV cell transformation vectors are particularly desirable because of their ability to be pseudotyped to infect non-dividing hematopoietic stem cells (CD34.sup.+). These stem cells differentiate into a variety of immune cells, including CD4.sup.+ cells which are the primary targets for HIV infection. CD34.sup.+ cells are the most important target cells for ex vivo gene therapy, because these cells differentiate into many different cell types, and because the cells are capable of re-engraftment into a patient undergoing ex vivo therapy.
Most of the previously reported HIV packaging systems have very low titers, ranging from 10.sup.0 to 10.sup.2 transducing units per ml (TU/ml), and/or result from transient transfections, which are unstable (Buchschacher, et al. (1992) J. Virol. 66(5):2731-2739; Rizvi, et al. (1993) J. Virol. 67(5):2681-2688; Carroll, et al. (1994 J. Virol. 68(9):6047-6051; Parolin, et al. (1994) J. Virol. 68(6):3888-3895; Shimada, et al. (1991) J. Clin. Invest. 88:10431047). A group reported a transduction efficiency of 2.times.10.sup.4 TU/ml, but in this study the packaging system was infectious wild type HIV-1 (Richardson, et al. (1993) J. Virol. 67(7):3997-4005), which is of little practical value. The only study describing a stable cell line doubly transfected with the packaging plasmid and the vector reported transduction lower than 10.sup.2 TU/ml (Carroll, et al. (1994) J. Virol. 68(9):6047-6051).
Thus, the efforts to develop HIV-based delivery systems have so far resulted in extremely low transduction efficiencies for HIV-based vectors (Buchschacher, et al. (1992) J. Virol. 66(5):2731-2739; Richardson, et al. (1993) J. Virol. 67(7):3997-4005; Rizvi, et al. (1993) J. Virol. 67(5):2681-2688; Carroll, et al. (1994 J. Virol. 68(9):6047-6051), rendering the use of HIV delivery systems impractical. This invention overcomes these and other problems. In addition, it is discovered that the regulatory elements which are used in HIV vectors (e.g. TAR, RRE and packaging signal sequences) are themselves antagonistic to HIV replication, thereby providing a level of HIV inhibition.